Use of creatine or creatine compounds for skin preservation

ABSTRACT

The present invention relates to the use of creatine compounds such as, for example, creatine, creatine phosphate or analogs of creatine, such as creatine-pyruvate, creatine-ascorbate, cyclocreatine, 3 guanidinopropionic acid, guanidinoacetate, homocyclocreatine, guanidino benzoates as energy generating systems and antioxidants for preservation of skin against adverse aging effects and damage secondary to insults such as harmful sun radiations, stress and fatigue. The creatine compounds which can be used in the present method include (1) creatine, creatine phosphate and analogs of these compounds which can act as substrates or substrate analogs for creatine kinase; (2) molecules that mimic the biological activity of creatine (3) molecules that modulate the creatine kinase system.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/088,519, filed on Mar. 23, 2005, which is a continuation of U.S. Ser.No. 09/852,966, filed on May 10, 2001, which is a continuation of U.S.Ser. No. 09/344,427, filed on Jun. 25, 1999 (now U.S. Pat. No. 6,242,491B1, issued on Jun. 5, 2001); the entire contents of each of theaforementioned applications are hereby incorporated herein by reference.

Creatine and creatine compounds are discussed in related applicationsincluding U.S. patent application Ser. No. 08/853,174, filed on May 7,1997; U.S. patent application Ser. No. 08/914,887, filed on Aug. 19,1997; U.S. patent application Ser. No. 08/736,967, filed on Oct. 25,1996; and U.S. patent application Ser. No. 09/285,395, filed on Apr. 2,1999. The entire contents of each of the applications, including allreferences cited therein, and hereby expressly incorporated herein byreference.

BACKGROUND OF THE INVENTION

The creatine kinase/creatine phosphate energy system is only onecomponent of an elaborate energy-generating system found in tissue withhigh and fluctuating energy requirements. The components of the creatineenergy system include the enzyme creatine kinase, the substratescreatine and creatine phosphate, and the transporter of creatine. Thereaction catalyzed by creatine kinase is: MgADP+PCr⁼+H⁺ MgATP⁼+Cr. Someof the functions associated with this system include efficientregeneration of energy in cells with fluctuating and high energydemands, energy transport to different parts of the cell, phosphoryltransfer activity, ion transport regulation, and involvement in signaltransduction pathways.

Creatine is a compound which is naturally occurring and is found inmammalian brain and other excitable tissues, such as skeletal muscle,retina and heart. Its phosphorylated form, creatine phosphate, also isfound in the same organs and is the product of the creatine kinasereaction utilizing creatine as a substrate. Creatine phosphate is one ofthe highest energy generating compounds in the cell and creatine is anexcellent stimulant of oxidative phosphorylation and high energyproduction. Creatine has been extensively used by body builders as ameans of stimulating energy production in the skeletal muscle. Creatineand creatine phosphate can be synthesized relatively easily and arebelieved to be non-toxic to mammals. Creatine, creatine phosphate andthe enzymes that utilize them as substrates, i.e. the creatine kinasesrepresent an efficient system for the rapid regeneration of energy.Kaddurah-Daouk et al. (WO 92/08456 published May 29, 1992 and WO90/09192, published Aug. 23, 1990; U.S. Pat. No. 5,321,030; and U.S.Pat. No. 5,324,731) describe methods of inhibiting the growth,transformation and/or metastasis of mammalian cells using relatedcompounds. Examples of compounds described by Kaddurah-Daouk et al.include cyclocreatine, b-guandidino propionic acid, homocyclocreatine,1-carboxymethyl-2-iminohexahydropyrimidine, guanidino acetate andcarbocreatine. These same inventors have also demonstrated the efficacyof such compounds for combating viral infections (U.S. Pat. No.5,321,030). Elebaly in U.S. Pat. No. 5,091,404 discloses the use ofcyclocreatine for restoring functionality in muscle tissue. Cohn in PCTpublication No. WO94/16687 described a method for inhibiting the growthof several tumors using creatine and related compounds. Kaddurah-Daouket. al. (WO 96/14063) reported on the neuroprotective effect of creatinecompounds especially against neurodegenerative diseases such asHuntington's, Parkinson's, ALS, Alzheimer's.

Aging involves death of cells or cell dysfunction due to production offree radicals, oxidative damage and energy depletion due tomitochondrial dysfunction. Harman (1988) linked senescence or death tothe injurious effects of free radicals arising from the one-electronreduction of oxygen during metabolism. There has been an inverserelationship between auto-oxidation rate in different animal species andlife expectancy in the same species (Cutler 1985; Sohal 1995).Mitochondria are the major source of oxygen radicals through therespiratory chain and are also deeply affected by reactive oxygenspecies (ROS), resulting in serious risks to their function.Mitochondrial dysfunction could result in defects in electron transport,oxidative phosphorylation and energy production resulting in cell damageand ultimately cell death.

SUMMARY OF THE INVENTION

The present invention relates to methods for protecting skin tissueagainst age related damage or insults such as harmful UV radiation,stress and fatigue by preserving energy pools and protecting againstfree radical production and oxidative stress. This is achieved byadministering an amount of a creatine compound or compounds whichmodulate one or more of the structural or functional components of thecreatine kinase/phosphocreatine system sufficient to prevent, reduce orameliorate skin damage or skin aging. Compounds which are effective forthis purpose include the natural compound creatine in its differenthydration or salt forms and analogs of creatine. The compounds can bemixed in with creams, oils, emulsions and the like to be spread readilyon skin surfaces. Alternatively, the compounds also can be packaged in asupplement form.

The present invention also provides compositions containing creatinecompounds in combination with a pharmaceutically or cosmeticallyacceptable carrier, and effective amounts of other agents which act ontissue preservation such as antioxidants (e.g., CoQ10), vitamins such asC, B5, B6, B9, E, energy enhancing agents (for example ATP, pyruvate,nicotinamide) and skin softeners to slow the process of aging.

Packaged formulations for preventing or slowing the process of agingalso are the subject of the present invention. The packaged formulationsinclude a container holding the creatine compound, in combination with apharmaceutically or cosmetically acceptable carrier, along withinstructions for administering the same for the purpose of preventing,ameliorating, arresting or eliminating the process of skin aging or skindamage.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 depicts the effects of 1% creatine supplementation on3-nitrotyrosine/tyrosine concentration in FALS mice. Animals were placedon 1% creatine at age 70 days and levels of 3-nitrotyrosine/tyrosineconcentrations were determined at age 120 days. White column representswild type mice, black G93A transgenic mice fed a normal diet and thedark gray column represents G93A transgenic mice fed a 1% creatine diet.*p is less than 0.05 compared with littermate controls; #p less than0.05 compared with creatine supplementation.

FIG. 2 depicts the effect of 1% creatine supplementation on hydroxylradical production as measured by rate of conversion of salicylate toits by products in FALS mice (G93A transgenic mice) after systemicadministration of mitochondrial toxin 3-NP. Administration of 3-NPresulted in a significant increase in the ratio of 3,4DHBA to 4HBA inmice fed normal diets, which was significantly attenuated in micereceiving 1% creatine supplementation. Black column represents basallevels, white after 3-NP administration. *p less than 0.05, comparedwith basal levels; #p less than 0.05 compared with mice treated with3-NP and fed a control diet.

FIG. 3 depicts the production of 2,3 and 2,5 DHBA and 3 nitrotyrosineafter intrastriatal injection of malonate in control animals fed withcreatine and those fed with cyclocreatine. **p is less than 0.01compared with uninjected side; #p is less than 0.05 compared with DHBAelevation in controls. Eight animals were used in each group.

FIG. 4 depicts 3-Nitrotyrosine levels after systemic administration ofthe mitochondrial toxin 3-NP to controls and creatine-fed animals. **pis less than 0.01 compared with saline controls. ##p is less than 0.01compared with 3-NP injected animals on a normal diet. Ten animals wereused in each group.

DETAILED DESCRIPTION OF THE INVENTION

The methods of the present invention generally comprise administering toa subject an amount of a creatine compound or compounds. It is thoughtthat the creatine compounds modulate one or more of the structural orfunctional components of the creatine kinase/phosphocreatine systemsufficient to prevent, reduce or ameliorate symptoms of aging and damageto the skin. Components of the system which can be modulated include theenzyme creatine kinase, the substrates creatine and creatine phosphate,and the transporter of creatine. The term “modulate,” “modulation” or“modulating” includes any increase or decrease in the activity of anycomponent of the creatine kinase/phosphocreatine system.

In one embodiment, the invention pertains to a method for treating asubject (e.g., a mammal, preferably, a human) for skin disorders byadministering to the subject an effective amount of a creatine compoundsuch that the skin damage is treated.

The term “mammal” includes any animal which may be treated by thecompositions of the invention. Examples of mammals include dogs, cats,horses, pigs, cows, rodents, horses, bears, monkeys, gorillas,chimpanzees, and, preferably, humans.

Creatine compounds are predicted to preserve tissue by boosting upenergy reserves in the skin and also by arresting mechanisms involved inoxidative damage and cell death. Compounds which are particularlyeffective for this purpose include creatine, creatine phosphate, andanalogs thereof which are described in detail below. The term “creatinecompounds” includes creatine, creatine phosphate, and compounds whichare structurally similar to creatine or creatine phosphate, and analogsof creatine and creatine phosphate. The term “creatine compounds” alsoincludes compounds which “mimic” the activity of creatine, creatinephosphate or creatine analogs. The term “mimics” is intended to includecompounds which may not be structurally similar to creatine but mimicthe therapeutic activity of creatine, creatine phosphate or structurallysimilar compounds. Also the term creatine compound includes “modulatorsof the creatine kinase system,” for example, compounds which modulatethe activity of the enzyme, or the activity of the transporter ofcreatine or the ability of other proteins or enzymes or lipids tointeract with the system.

The term “treatment” includes the diminishment or alleviation of atleast one symptom associated or caused by the disorder being treated.For example, treatment can be diminishment of several symptoms of adisorder or complete eradication of a disorder.

The language “treating for skin disorders” includes both prevention ofdisorders, amelioration and/or arrest of the disorder process. Examplesof skin disorders include, but are not limited to aging and damageresulting from sun radiation, stress, fatigue and/or free radicals.Although not wishing to be bound by theory, the creatine compoundsdescribed herein are thought to have both curative and prophylaceticeffects on development of damage and aging of the skin and other tissue.The language also includes any amerlioration or arrest of any symptomsassociated with the disorder process (e.g., wrinkles). For example,treating wrinkles may include preventing, retarding, arresting, orreversing the process of wrinkle formation in skin, e.g., mammalianskin, preferably, human skin.

In a further embodiment, the method includes coadministration of thecreatine compound with a pharmaceutically acceptable carrier.Preferably, the pharmaceutically acceptable carrier is suitable fortopical administration.

The term “topical administration” includes methods of delivery such aslaying on or spreading on the skin. It involves any form ofadministration which involves the skin. Examples of compositionssuitable for topical administration, include but are not limited to,ointments, lotions, creams, cosmetic formulations, and skin cleansingformulations. Additional examples include aerosols, solids (such as barsoaps) and gels.

The term “pharmaceutically acceptable” includes drugs, medicaments orinert ingredients which are suitable for use in contact with the tissuesof humans and lower animals without undue toxicity, incompatibility,instability, irritation, allergic response, and the like, commensuratewith a reasonable benefit/risk ratio. The term also encompassescosmetically acceptable ingredients.

The language “therapeutically or cosmetically effective amount” isintended to include the amount of the creatine compound sufficient toprevent onset of aging or damage to the skin or significantly reduceprogression of damage in the subject being treated. A therapeutically orcosmetically effective amount can be determined on an individual basisand will be based, at least in part, on consideration of the severity ofthe symptoms to be treated and the activity of the specific analogselected if an analog is being used. Further, the effective amounts ofthe creatine compound may vary according to the age of the subject beingtreated. Thus, a therapeutically or cosmetically effective amount of thecreatine compound can be determined by one of ordinary skill in the artemploying such factors as described above using no more than routineexperimentation in health care management.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting a compound(s) of thepresent invention within or to the subject such that it can performs itsintended function. Typically, such compounds are carried or transportedfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid; fruitacids, pyrogen-free water; isotonic saline; Ringer's solution; ethylalcohol; phosphate buffer solutions; and other non-toxic compatiblesubstances employed in pharmaceutical formulations.

The topical pharmaceutical compositions of the present invention may bemade into a wide variety of product types. These include, but are notlimited to solutions, lotions, creams, beach products, gels, sticks,sprays, pads, ointments, pastes, mousses and cosmetics. These producttypes may comprise several types of carrier systems including, but notlimited to solutions, emulsions, gels and solids.

The topical pharmaceutical compositions of the present inventionformulated as solutions typically include a pharmaceutically-acceptableaqueous or organic solvent. The terms “pharmaceutically-acceptableaqueous solvent” and “pharmaceutically-acceptable organic solvent” referto a solvent which is capable of having dispersed or dissolved thereinthe active compound, and possesses acceptable safety properties (e.g.,irritation and sensitization characteristics). Water is a typicalaqueous solvent. Examples of suitable organic solvents include:propylene glycol, butylene glycol, polyethylene glycol (200-600),polypropylene glycol (425-2025), glycerol, 1,2,4-butanetriol, sorbitolesters, 1,2,-6-hexanetriol, ethanol, isopropanol, butanediol, andmixtures thereof. Preferably, these solutions contain from about 0.01%to about 50% of the active compound, more preferably from about 0.1% toabout 20%; and from about 1% to about 80% of an acceptable aqueous ororganic solvent, more preferably from about 1% to about 40%.

If the topical pharmaceutical compositions of the present invention areformulated as an aerosol and applied to the skin as a spray-on, apropellant is added to a solution composition. A more completedisclosure of propellants useful herein can be found in Sagarin,Cosmetics Science and Technology, 2nd Edition, Vol. 2, pp. 443-465(1972).

Topical pharmaceutical compositions of the present invention may beformulated as a solution comprising an emollient. An example of acomposition formulated in this way would be a sunscreen-containingproduct. Preferably, such compositions contain from about 0.1% to about50% of the active compound and from about 2% to about 50% of a topicalpharmaceutically-acceptable emollient.

As used herein, “emollients” refer to materials used for the preventionor relief of dryness, as well as for the protection of the skin. A widevariety of suitable emollients are known and may be used herein.Sagarin, Cosmetics, Science and Technology, 2nd Edition, Vol. 1, pp.32-43 (1972), incorporated herein by reference, contains numerousexamples of suitable materials.

A lotion can be made from a solution carrier system. Lotions preferablycomprise from about 0.1% to about 20%, more preferably from about 1% toabout 5%, of the active compound; from about 1% to about 20%, preferablyfrom about 5% to about 10%, of an emollient; and from about 50% to about90%, preferably from about 60% to about 80%, water.

Another type of product that may be formulated from a solution carriersystem is a cream. A cream of the present invention would preferablycomprise from about 0.1% to about 20%, more preferably from about 1% toabout 5%, of the active compound; from about 5% to about 50%, preferablyfrom about 10% to about 20%, of an emollient, and from about 45% toabout 85%, preferably from about 50% to about 75%, water.

Yet another type of product that may be formulated from a solutioncarrier system is an ointment. An ointment may comprise a simple base ofanimal or vegetable oils or semi-solid hydrocarbons (oleaginous).Ointments may also comprise absorption ointment bases which absorb waterto form emulsions. Ointment carriers may also be water soluble. Anointment may also comprise from about 2% to about 10% of an emollientplus from about 0.1% to about 2% of a thickening agent. A more completedisclosure of thickening agents useful herein can be found in Segarin,Cosmetics, Science and Technology, 2nd Edition, Vol. 1, pp. 72-73(1972).

If the carrier is formulated as an emulsion, from about 1% to about 10%,preferably from about 2% to about 5%, of the carrier system comprises anemulsifier. Emulsifiers may be nonionic, anionic or cationic. Suitableemulsifiers are disclosed in, for example, U.S. Pat. No. 3,755,560,issued Aug. 28, 1973, Dickert et al.; U.S. Pat. No. 4,421,769, issuedDec. 20, 1983, Dixon et al.; and McCutcheon's Detergents andEmulsifiers, North American Edition, pages 317-324 (1986); thedisclosures of which are incorporated herein by reference. Preferredemulsifiers are anionic or nonionic, although the other types may alsobe used.

Lotions and creams can be formulated as emulsions as well as solutions.Preferably such lotions comprise from about 0.1% to about 20%, morepreferably from about 1% to about 5%, of the active compound; from about1% to about 20%, preferably from about 5% to about 10%, of an emollient;from about 25% to about 75%, preferably from about 45% to about 95%,water; and from about 0.1% to about 10%, preferably from about 0.5% toabout 5%, of an emulsifier. Such creams would preferably comprise fromabout 0.1% to about 20%, more preferably from about 1% to about 5%, ofthe active compound; from about 1% to about 20%, preferably from about5% to about 10%, of an emollient; from about 20% to about 80%,preferably from about 30% to about 70%, water; and from about 1% toabout 10%, preferably from about 2% to about 5%, of an emulsifier.

Single emulsion skin care preparations, such as lotions and creams, ofthe oil-in-water type and water-in-oil type are well-known in thecosmetic art and are useful in the present invention. Multiphaseemulsion compositions, such as the water-in-oil-in-water type, asdisclosed in U.S. Pat. No. 4,254,105, Fakuda et al., issued Mar. 3,1981, incorporated herein by reference, are also useful in the presentinvention. In general, such single or multiphase emulsions containwater, emollients and emulsifiers as essential ingredients.

Triple emulsion carrier systems comprising an oil-in-water-in-siliconefluid emulsion composition as disclosed in U.S. Pat. No. 4,960,764,Figueroa, issued Oct. 2, 1990, are also useful in the present invention.Preferably, this triple emulsion carrier system can be combined withfrom about 0.1% to about 20%, more preferably from about 1% to about 5%,of the active compound to yield the topical pharmaceutical compositionof the present invention.

Another emulsion carrier system useful in the topical pharmaceuticalcompositions of the present invention is a micro-emulsion carriersystem. Such a system comprises from about 9% to about 15% squalane;from about 25% to about 40% silicone oil; from about 8% to about 20% ofa fatty alcohol; from about 15% to about 30% of polyoxyethylene sorbitanmono-fatty acid (commercially available under the trade name Tweens) orother nonionics; and from about 7% to about 20% water. This carriersystem is preferably combined with from about 1% to about 5% of theactive compound.

If the topical pharmaceutical compositions of the present invention areformulated as a gel or a cosmetic stick, a suitable amount of athickening agent, as disclosed supra, is added to a cream or lotionformulation.

The topical pharmaceutical compositions of the present invention mayalso be formulated as makeup products such as foundations.

The topical pharmaceutical compositions of the present invention mayalso be formulated as medicated pads. Suitable examples of these padsare fully disclosed in U.S. Pat. Nos. 4,891,227 and 4,891,228, to Thamanet al., both issued Jan. 2, 1990 the disclosures of which areincorporated herein.

The topical pharmaceutical compositions of the present invention maycontain, in addition to the aforementioned components, a wide variety ofadditional oil-soluble materials and/or water-soluble materialsconventionally used in topical compositions, at their art-establishedlevels.

Various water-soluble materials may also be present in the compositionsof this invention. These include humectants, proteins and polypeptides,preservatives and an alkaline agent. In addition, the topicalcompositions herein can contain conventional cosmetic adjuvants, such asdyes, opacifiers (e.g., titanium dioxide), pigments and perfumes.

The topical pharmaceutical compositions of the present invention mayalso include a safe and effective amount of a penetration enhancingagent. A preferred amount of penetration enhancing agent is from about1% to about 5% of the composition. Another useful penetration enhancerfor the present invention is the non-ionic polymer under the CTFAdesignation: polyacrylamide and isoparrafin and laureth-7, available asSepigel from Seppic Corporation. Also useful is polyquaternium-32 andmineral oil known as SalCare SC92 available from Allied Colloids,Suffolk, Va. This is a class of cationic polymers which are generallydescribed in U.S. Pat. No. 4,628,078 to Glover et al. issued Dec. 9,1986 and U.S. Pat. No. 4,599,379 to Flesher et al. issued Jul. 8, 1986both of which are incorporated by reference herein.

Examples of useful penetration enhancers, among others, are disclosed inU.S. Pat. Nos. 4,537,776, Cooper, issued Aug. 27, 1985; 4,552,872,Cooper et al., issued Nov. 12, 1985; 4,557,934, Cooper, issued Dec. 10,1985; 4,130,667, Smith, issued Dec. 19, 1978; 3,989,816, Rhaadhyaksha,issued Nov. 2, 1976; 4,017,641, DiGiulio, issued Apr. 12, 1977; andEuropean Patent Application 0043738, Cooper et al., published Jan. 13,1982.

Other conventional skin care product additives may also be included inthe compositions of the present invention. For example, collagen,hyaluronic acid, elastin, hydrolysates, primrose oil, jojoba oil,epidermal growth factor, soybean saponins, mucopolysaccharides, andmixtures thereof may be used.

Various vitamins may also be included in the compositions of the presentinvention. For example, Vitamin A, ascorbic acid, Vitamin B, biotin,panthothenic acid, Vitamin D, Vitamin E and mixtures thereof andderivatives thereof are contemplated.

Also contemplated are skin cleaning compositions comprising both activecompounds of the present invention and a cosmetically-acceptablesurfactant. The term “cosmetically-acceptable surfactant” refers to asurfactant which is not only an effective skin cleanser, but also can beused without undue toxicity, irritation, allergic response, and thelike. Furthermore, the surfactant must be capable of being commingledwith the active compound in a manner such that there is no interactionwhich would substantially reduce the efficacy of the composition forregulating skin damage, e.g., wrinkles.

The skin cleaning compositions of the present invention preferablycontain from about 0.1% to about 20%, preferably from about 1% to about5%, of the creatine compound (e.g., creatine, cyclocreatine or anothercreatine compound) and from about 1% to about 90%, more preferably fromabout 1% to about 10%, of a cosmetically-acceptable surfactant.

The physical form of the skin cleansing compositions is not critical.The compositions can be, for example, formulated as toilet bars,liquids, pastes, mousses, or pads.

The surfactant component of the compositions of the present inventionare selected from anionic, nonionic, zwitterionic, amphoteric andampholytic surfactants, as well as mixtures of these surfactants. Suchsurfactants are well-known to those skilled in the detergency art.

The cleaning compositions of the present invention can optionallycontain, at their art-established levels, materials which areconventionally used in skin cleansing compositions.

Sunblocks and sunscreens incorporating creatine compounds are alsocontemplated. The term “sun block” or “sun screen” includes compositionswhich block UV light. Examples of sunblocks include, for example, zincoxide and titanium dioxide.

Sun radiation is one cause major cause of skin damage, e.g., wrinkles.Thus, for purposes of wrinkle treatment or prevention, the combinationof creatine compounds with a UVA and/or UVB sunscreen would beadvantageous. The inclusion of sunscreens in compositions of the presentinvention will provide immediate protection against acute UV damage.Thus, the sunscreen will prevent further skin damage caused by UVradiation, while the compounds of the invention regulates existing skindamage.

A wide variety of conventional sunscreening agents are suitable for usein combination with the active compound. Segarin, et al., at ChapterVIII, pages 189 et seq., of Cosmetics Science and Technology, disclosenumerous suitable agents. Specific suitable sunscreening agents include,for example: p-aminobenzoic acid, its salts and its derivatives (ethyl,isobutyl, glyceryl esters; p-dimethylaminobenzoic acid); anthranilates(i.e., o-aminobenzoates; methyl, menthyl, phenyl, benzyl, phenylethyl,linalyl, terpinyl, and cyclohexenyl esters); salicylates (amyl, phenyl,benzyl, menthyl, glyceryl, and dipropyleneglycol esters); cinnamic acidderivatives (methyl and benzyl esters, alpha.-phenyl cinnamonitrile;butyl cinnamoyl pyruvate); Dihydroxycinnamic acid derivatives(umbelliferone, methylumbelliferone, methylaceto-umbelliferone);trihydroxycinnamic acid derivatives (esculetin, methylesculetin,daphnetin, and the glucosides, esculin and daphnin); hydrocarbons(diphenylbutadiene, stilbene); dibenzalacetone and benzalacetophenone;Naphtholsulfonates (sodium salts of 2-naphthol-3,6-disulfonic and of2-naphthol-6,8-disulfonic acids); Dihydroxy-naphthoic acid and itssalts; o- and p-Hydroxybiphenyldisulfonates; Coumarin derivatives(7-hydroxy, 7-methyl, 3-phenyl); Diazoles (2-acetyl-3-bromoindazole,phenyl benzoxazole, methyl naphthoxazole, various aryl benzothiazoles);Quinine salts (bisulfate, sulfate, chloride, oleate, and tannate);Quinoline derivatives (8-hydroxyquinoline salts, 2-phenylquinoline);Hydroxy- or methoxy-substituted benzophenones; Uric and vilouric acids;Tannic acid and its derivatives (e.g., hexaethylether); (Butyl carbotol)(6-propyl piperonyl) ether; Hydroquinone; Benzophenones (Oxybenzene,Sulisobenzone, Dioxybenzone, Benzoresorcinol,2,2′,4,4′-Tetrahydroxybenzophenone,2,2′-Dihydroxy-4,4′-dimethoxybenzophenone, Octabenzone;4-Iso-propyldibenzoylmethane; Butylmethoxydibenzoylmethane; Etocrylene;and 4-isopropyl-di-benzoylmethane.

Preferred sunscreens useful in the compositions of the present inventionare 2-ethylhexyl-p-methoxycinnamate, butylmethoxydibenzoylmethane,2-hydroxy-4-methoxybenzophenone, octyldimethyl-p-aminobenzoic acid andmixtures thereof.

A safe and effective amount of sunscreen may be used in the compositionsof the present invention. The sunscreening agent must be compatible withthe active compound. Generally the composition may comprise from about1% to about 20%, preferably from about 2% to about 10%, of asunscreening agent. Exact amounts will vary depending upon the sunscreenchosen and the desired Sun Protection Factor (SPF).

Also particularly useful in the present invention are sunscreens such asthose disclosed in Sabatelli, U.S. patent application Ser. No. 054,085(filed Jun. 2, 1987) and Sabatelli et al., U.S. patent application Ser.No 054,046 (filed Jun. 2, 1987). The sunscreening agents disclosedtherein have, in a single molecule, two distinct chromophore moietieswhich exhibit different ultra-violet radiation absorption spectra. Oneof the chromophore moieties absorbs predominantly in the UVB radiationrange and the other absorbs strongly in the UVA radiation range.

An agent may also be added to any of the compositions of the presentinvention to improve the skin substantivity of those compositions,particularly to enhance their resistance to being washed off by water,or rubbed off. A preferred agent which will provide this benefit is acopolymer of ethylene and acrylic acid. Compositions comprising thiscopolymer are disclosed in U.S. Pat. No. 4,663,157, Brock, issued May 5,1987, which is incorporated herein by reference.

In another embodiment of the present invention, an anti-inflammatoryagent is included as an active agent along with the creatine compoundsof the invention. The anti-inflammatory agent protects strongly in theUVA radiation range (though it also provides some UVB protection aswell) thereby preventing further skin damage caused by UV radiation,while the creatine compounds of the invention treats existing damage.Thus the combination provides broad protection. The topical use ofanti-inflammatory agents reduces photo-aging of the skin resulting fromchronic exposure to UV radiation. (See U.S. Pat. No. 4,847,071, Bissett,Bush, and Chatterjee, issued Jul. 11, 1989, incorporated herein byreference; and U.S. Pat. No. 4,847,069, Bissett and Chatterjee, issuedJul. 11, 1989, incorporated herein by reference.)

A safe and effective amount of an anti-inflammatory agent may be addedto the compositions of the present invention, preferably from about 0.1%to about 10%, more preferably from about 0.5% to about 5%, of thecomposition. The exact amount of anti-inflammatory agent to be used inthe compositions will depend on the particular anti-inflammatory agentutilized since such agents vary widely in potency.

Steroidal anti-inflammatory agents, including but not limited to,corticosteroids such as hydrocortisone, hydroxyltriamcinolone,alpha-methyl dexamethasone, dexamethasone-phosphate, beclomethasonedipropionate, clobetasol valerate, desonide, desoxymethasone,desoxycorticosterone acetate, dexamethasone, dichlorisone, diflorasonediacetate, diflucortolone valerate, fluadrenolone, flucloroloneacetonide, fludrocortisone, flumethasone pivalate, fluosinoloneacetonide, fluocinonide, flucortine butylester, fluocortolone,fluprednidene (fluprednylidene) acetate, flurandrenolone, halcinonide,hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone,triamcinolone acetonide, cortisone, cortodoxone, flucetonide,fludrocortisone, difluorosone diacetate, fluradrenolone acetonide,medrysone, amcinafel, amcinafide, betamethasone and the balance of itsesters, chloroprednisone, chlorprednisone acetate, clocortelone,clescinolone, dichlorisone, difluprednate, flucloronide, flunisolide,fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate,hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone,paramethasone, prednisolone, prednisone, beclomethasone dipropionate,triamcinolone, and mixtures thereof may be used.

A second class of anti-inflammatory agents which is useful in thecompositions of the present invention includes the nonsteroidalanti-inflammatory agents. The variety of compounds encompassed by thisgroup are well-known to those skilled in the art. For detaileddisclosure of the chemical structure, synthesis, side effects, etc., ofnon-steroidal anti-inflammatory agents, reference may be had to standardtexts, including Antiinflammatory and Anti-Rheumatic Drugs, K. D.Rainsford, Vol. 1-III, CRC Press, Boca Raton, (1985), andAnti-inflammatory Agents. Chemistry and Pharmacology, 1, R. A. Scherrer,et al., Academic Press, New York (1974). Mixtures of these non-steroidalanti-inflammatory agents may also be employed, as well as thepharmaceutically-acceptable salts and esters of these agents. Forexample, etofenamate, a flufenamic acid derivative, is particularlyuseful for topical application. Yet another class of anti-inflammatoryagents which are useful in the present invention are those disclosed inU.S. Pat. No. 4,912,248, Maeller, issued Mar. 27, 1990. This patentdiscloses compounds and diastereomeric mixtures of specific2-naphthyl-containing ester compounds, especially naproxen ester andnaproxol ester compounds, having two or more chiral centers. Finally,so-called “natural” anti-inflammatory agents are useful in the presentinvention. For example, candelilla wax, alpha bisabolol, aloe vera,Manjistha (extracted from plants in the genus Rubia, particularly RubiaCordifolia), and Guggal (extracted from plants in the genus Commiphora,particularly Commiphora Mukul), may be used.

In another embodiment, the skin composition further comprises a safe andeffective amount of a skin protectant. The skin protectant preferablycomprises from about 0.001% to about 2%, more preferably from about0.01% to about 1% of the composition. Useful skin protectants aredisclosed in the Federal Register Vol. 48, No. 32 and include allantoin,aluminum hydroxide gel, bismuth subnitrate, boric acid, calamine, cocoabutter, corn starch, dimethicone, glycerin, kaolin, live yeast cellderivative, petrolatum, shark liver oil, sodium bicarbonate, sulfur,tannic acid, white petrolatum, zinc acetate, zinc carbonate and zincoxide and mixtures thereof.

As set out above, certain embodiments of the present compounds cancontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable acids. The term “pharmaceutically acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ during the final isolation andpurification of the compounds of the invention, or by separatelyreacting a purified compound of the invention in its free base form witha suitable organic or inorganic acid, and isolating the salt thusformed. Representative salts include the hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci.66:1-19).

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ during the final isolation and purification of thecompounds, or by separately reacting the purified compound in its freeacid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like.

The term “pharmaceutically acceptable esters” refers to the relativelynon-toxic, esterified products of the compounds of the presentinvention. These esters can be prepared in situ during the finalisolation and purification of the compounds, or by separately reactingthe purified compound in its free acid form or hydroxyl with a suitableesterifying agent. Carboxylic acids can be converted into esters viatreatment with an alcohol in the presence of a catalyst. Hydroxyls canbe converted into esters via treatment with an esterifying agent such asalkanoyl halides. The term is further intended to include lowerhydrocarbon groups capable of being solvated under physiologicalconditions, e.g., alkyl esters, methyl, ethyl and propyl esters. (See,for example, Berge et al., supra.)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Formulations of the present invention include those suitable fortopical, oral, nasal, transdermal, buccal, sublingual, rectal, vaginaland/or parenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willgenerally be that amount of the compound which produces a therapeuticeffect. Generally, out of one hundred percent, this amount will rangefrom about 1 percent to about ninety-nine percent of active ingredient,preferably from about 5 percent to about 70 percent, most preferablyfrom about 10 percent to about 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the activecompound in a polymer matrix or gel.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

The preparations of the present invention may be given parenterally,topically, or rectally. They are of course given by forms suitable foreach administration route. For example, they are administered in tabletsor capsule form, by injection, inhalation, eye lotion, ointment,suppository, etc. administration by injection, infusion or inhalation;topical by lotion or ointment, and rectal by suppositories. Topicaladministration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systematically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous andsubcutaneous doses of the compounds of this invention for a patient,when used for the indicated effects, will range from about 0.0001 toabout 1000 mg per kilogram of body weight per day. Creatine has beentaken by athletes in the range of 2-30 gms per day with reasonablesafety profile. Topical creams with 0.01-10% creatine are anticipated tobe well tolerated. If desired, the effective daily dose of the activecompound may be administered as two, three, four, five, six or moresub-doses administered separately at appropriate intervals throughoutthe day, optionally, in unit dosage forms.

The term “chronic treatment” includes continued treatment with acreatine compound over an extended period during a subject's lifetime,preferably for at least about three weeks, more preferably from aboutthree months to about twenty years, more preferably from about sixmonths to about ten years, more preferably still from about one year toabout five years.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical composition.

In a further embodiment, the skin disorder is associated with freeradicals, aging, sun radiation, stress or fatigue. In anotherembodiment, the subject is afflicted with wrinkles or is at risk for askin disorder.

The term “associated with free radicals” includes any disorders ordamaged to the skin resulting from, directly or indirectly from freeradicals. The free radicals may be initiated by, for example, sunradiation (e.g., UV radiation) or pollution.

The term “aging” includes processes where there is oxidative damage,energy depletion or mitochondrial dysfunction where onset, amelioration,arrest, or elimination is effectuated by the creatine compoundsdescribed herein. Symptoms of aging include, but are not limited to,wrinkles, loss of elasticity of the skin and uneven pigmentation of theskin.

The term “subject” includes living organisms susceptible to skin damageor aging. Examples of subjects include humans, dogs, cats, horses,goats, cows, cattle. The term “subject” further is intended to includetransgenic species.

The invention also features a composition for the treatment of the skinof a subject. The composition comprises an effective amount of creatine,creatine phosphate, a creatine compound or a salt thereof, and apharmaceutically acceptable carrier. Preferably, the effective amount iseffective to treat or prevent a skin disorder. Preferably, thecomposition is suitable for topical administration. The composition maybe formulated as a lotion, cream, or ointment, gel or solid. In oneadvantageous embodiment, the composition also contains a sunblock orsunscreen (e.g., zinc oxide or titanium dioxide). In another furtherembodiment, the composition may be formulated as a cosmetic foundationor as a skin cleansing agent. Advantageously, the composition maycontain a penetration agent. Examples of compounds which may beincorporated into the composition of the invention include, but are notlimited to, hydroxyacids, retinols, Aloe, Chamomile, or mixturesthereof. In a further embodiment, the skin disorder is associated withfree-radicals, aging, sun radiation, stress or fatigue.

In a further embodiment, the invention contemplates co-administering tothe subject an effective amount of a skin preserving agent. Examples ofskin preserving agents include antioxidants, such as ascorbic acid,vitamins, coenzyme Q10 (CoQ10) and its derivatives, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.Preferred anti-oxidants include, CoQ10 and vitamin E. Other examples ofskin preserving agents include energy-enhancing agents (e.g., ATP,nicotinamide or pyruvate), vitamins (e.g., E, C, B5, B6, and B9) andvitamin precursors.

The term “energy enhancing agents” also includes stimulants ofmitochondrial function or ATP production elsewhere in the cell. Examplesinclude intermediates such as, for example, pyruvate, nicotinamide andCoQ10.

Aging Oxidative Stress and Mitochondrial Dysfunction:

A common feature of the life cycle of virtually all multicellularorganisms is the progressive decline in efficiency of variousphysiological processes once the productive phase of life is over. Datahas supported the hypothesis that senescence cell death secondary toloss of functional capacity is due to accumulation of molecularoxidative damage (Harman 1956; Stadtman 1992; Ames et. al., 1993; Sohal1995). The hypothesis is based on the fact that oxygen is potentially atoxic substance, and its use by aerobes, although necessary for theirimmediate survival, also may be hazardous to their long term existence.Molecular oxygen is the precursor of superoxide, hydrogen peroxide andhydroxyl radicals. Upon further reactions these could generate reactiveoxygen species that cause extensive oxidative damage to macromolecules.Lipid peroxidation, DNA damage and carbonylation of proteins are some ofthe devastating effects. During aging there is an increase in the amountof oxidative stress which could be a result of increase in the rate ofgeneration of reactive oxygen species, or the decline in anti-oxidativedefenses or the decline in the efficiency of repair or removal ofdamaged molecules (Sohal et. al., 1996). With aging there is an increasein the production of ROS from mitochondria which results in damage tothe inner mitochondrial membrane. By positive feedback mechanisms thisresults in further increase in ROS. Among flies, those with a longerlife expectancy were shown to exhibit a lower rate of mitochondrialsuperoxide, hydrogen peroxide generation, a lower rate of proteinoxidative damage, less DNA oxidative damage, higher activities of SODand catalase, increased glutathione a versatile intracellular reductant.Variations in maximum life span among different species are oftenassociated with differences in the metabolic rate (rate of oxygenconsumption), metabolic potential (total amount of energy consumed pergram of body weight during life span) and level of oxidative stress. Thehighest degree of oxidative damage occur in tissues such as brain, heartand skeletal muscle which are composed primarily of long livedpostmitotic cells. These tissues are also the targets of several agerelated degenerative disorders in which oxidative stress has beenimplicated (Davies 1995; Weindruch et al., 1993). Agents that minimizethe production of reactive oxygen species are predicted to beprotective.

Creatine Kinase Isoenzymes

Cells require energy to survive and to carry out the multitude of tasksthat characterize biological activity. Cellular energy demand and supplyare generally balanced and tightly regulated for economy and efficiencyof energy. Creatine kinase plays a key role in the energy metabolism ofcells with intermittently high and fluctuating energy requirements suchas skeletal and cardiac muscle, brain and neural tissues, including, forexample, the retina, spermatozoa and electrocytes use (Bessman 1985,Wallimann 1992, Wyss and Kaddurah-Daouk 1999). As stated above, theenzyme catalyzes the reversible transfer of the phosphoryl group fromcreatine phosphate to ADP, to generate ATP. There are multi-isoforms ofcreatine kinase (CK) which include muscle (CK-M), brain (CK-BB) andmitochondrial (CK-Mia, CK-Mib) isoforms. The mitochondrial creatinekinases are located mainly between the inner and outer mitochondrialmembranes where in association with the adenine nucleotide translocaseand other proteins can transform ATP to phosphocreatine and transport itto other sites in the cell for energy utilization. The mitochondrialoctameric creatine kinase has been implicated in mitochondrialpermeability transition and cell death processes (O'Gorman 1997).Creatine is an excellent stimulant of oxidative phosphorylation andseems to induce octameric mitochondrial creatine kinase formation andinhibits mitochondrial pore opening. The cytoplasmic isoforms utilizephosphocreatine to produce ATP for cell work. A communication betweenthe mitochondrial and cytoplasmic isoforms through the substratescreatine and phosphocreatine ensures connectivity between sites ofenergy production and sites of energy consumption.

Experimental data suggest that CK is located near the sites in cellswhere energy generation occurs; e.g. where force generation by motorproteins takes place, next to ion pumps and transporters in membranesand where other ATP-dependent processes take place. It seems to play acomplex multi-faceted role in cellular energy homeostasis. The creatinekinase system is involved in energy buffering/energy transportactivities. It also is involved in regulating ADP and ATP levelsintracellularly as well as ADP/ATP ratios. Proton buffering andproduction of inorganic phosphate are important parts of the system.

Creatine Kinase, Skin Aging and Skin Damage

The creatine content and the efficiency of the creatine kinase systemdecreases with aging. Aging and several insults result in oxidativestress state and energy compromise. It is demonstrate here thatmodulation of the creatine kinase system results in minimizing the rateof production of molecules associated with oxidative damage. Suchminimization combined with energy boosting effects should slow damage totissue during aging or exposure to insults. Creatine and analogs ofcreatine that modify the rate of ATP synthesis through creatine kinasecould sustain energy production, mitochondrial function, and protectagainst free radical production. Such effects could have positive impactagainst aging or insult related skin damage.

The components of the creatine kinase/phosphocreatine system include theenzyme creatine kinase, the substrates creatine and creatine phosphate,and the transporter of creatine. Some of the functions associated withthis system include efficient regeneration of energy in cells withfluctuating and high energy demand, phosphoryl transfer activity, iontransport regulation, cytoskeletal association, nucleotide poolpreservation, proton buffering, and involvement in signal transductionpathways.

Without wishing to be bound by theory, it is thought that modulating thecreatine kinase activity would modulate energy flow and affect skin cellfunction, integrity and survival. An activated energy state shouldminimizes oxidative damage and enable cells to withstand insultsecondary to aging or insults such as UV radiation.

Creatine is taken by athletes to boost muscle function during burstactivity (for review see Wyss and Kaddurah-Daouk 1999) and duringcompetitions. Creatine was shown to have neuroprotective properties inseveral animal models of neurodegenerative diseases (Matthews et al.,1988; Kliveny et al. 1999; Matthews et. al., 1999).

Ingestion of creatine analogs has been shown to result in replacement oftissue phosphocreatine pools by synthetic phosphagens with differentkinetic and thermodynamic properties. This results in subtle changes ofintracellular energy metabolism, including the increase of totalreserves of high energy phosphate (see refs. Roberts, J. J. and J. B.Walker, Arch Biochem. Biophys 220(2): 563-571 (1983)). The replacementof phosphocreatine pools with slower acting synthetic phosphagens, suchas creatine analogs might benefit neurological disorders by providing alonger lasting source of energy. One such analog, cyclocreatine(1-carboxymethyl-2-aminoimidazolidine) modifies the flow of energy ofcells in stress and may interfere with ATP utilization at sites ofcellular work.

Creatine Compounds Useful in Skin Care

Creatine compounds useful in the present invention include compoundswhich modulate one or more of the structural or functional components ofthe creatine kinase/phosphocreatine system. Compounds which areeffective for this purpose include creatine, creatine phosphate andanalogs thereof, compounds which mimic their activity, and salts ofthese compounds as defined above. Exemplary creatine compounds aredescribed below.

Creatine (also known as N-(aminoiminomethyl)-N-methylglycine;methylglycosamine or N-methyl-guamido acetic acid) is a well-knownsubstance. (See, The Merck Index, Eleventh Edition, No. 2570 (1989).

Creatine is phosphorylated chemically or enzymatically by creatinekinase to generate creatine phosphate, which also is well-known (see,The Merck Index, No. 7315). Both creatine and creatine phosphate(phosphocreatine) can be extracted from animal tissue or synthesizedchemically. Both are commercially available.

Cyclocreatine is an essentially planar cyclic analog of creatine.Although cyclocreatine is structurally similar to creatine, the twocompounds are distinguishable both kinetically and thermodynamically.Cyclocreatine is phosphorylated efficiently by creatine kinase in theforward reaction both in vitro and in vivo. Rowley, G. L., J. Am. Chem.Soc. 93: 5542-5551 (1971); McLaughlin, A. C. et. al., J. Biol. Chem.247, 4382-4388 (1972).

The phosphorylated compound phosphocyclocreatine is structurally similarto phosphocreatine; however, the phosphorous-nitrogen (P—N) bond ofcyclocreatine phosphate is more stable than that of phosphocreatine.LoPresti, P. and M. Cohn, Biochem. Biophys. Acta 998: 317-320 (1989);Annesley, T. M. and J. B. Walker, J. Biol. Chem. 253; 8120-8125, (1978);Annesley, T. M. and J. B. Walker, Biochem. Biophys. Res. Commun. 74:185-190 (1977).

3-Guanidinopropionic acid (3-GPA) is an endogenous metabolite found inanimals and humans (Hiraga et. al., J. of Chromatography vol 342,269-275, 1985; Watanabe et. al., Guanidines edited by Mori et. al.,Plenum, N.Y., 49-58, 1983). The compound is available from Sigmachemicals and is an extensively studied analog of creatine.

Guanidino acetate is yet another analog of creatine and is a precursorof creatine in its biosynthetic pathway.

Guanidino benzoic acids are structurally related to creatine. Alsocompounds that attach amino acid like molecules covalently to creatineare creatine compounds of interest. Examples are creatine-ascorbate andcreatine-pyruvate. Other types of molecules could be covalentlyattached.

Creatine analogs and other agents which act to interfere with theactivity of creatine biosynthetic enzymes or with the creatinetransporter are useful in the present method of treating or preventingage related damage. Thus the effects of such compounds can be direct orindirect, operating by mechanisms including, but not limited to,influencing the uptake or biosynthesis of creatine, the function of thecreatine phosphate shuttle, enzyme activity, or the activity ofassociated enzymes, or altering the levels of substrates or products ofa reaction to alter the velocity of the reaction.

Substances known or believed to modify energy production through thecreatine kinase/phosphocreatine system which can be used in the presentmethod are described below. Exemplary compounds are shown in Tables 1and 2.

It will be possible to modify the substances described below to produceanalogs which have enhanced characteristics, such as greater specificityfor the enzyme, enhanced stability, enhanced uptake into cells, orbetter binding activity.

Compounds which modify the structure or function of the creatinekinase/creatine phosphate system directly or indirectly are useful inpreventing and/or treating age related damage to tissue such as skin.

Molecules that regulate the transporter of creatine, or the associationof creatine kinase with other protein or lipid molecules in themembrane, the substrates concentration creatine and creatine phosphatealso are useful in preventing and/or treating age related damage totissue such as skin.

Compounds which are useful in the present invention can be substrates,enzyme activity modifiers or substrate analogs of creatine kinase. Inaddition, modulators of the enzymes that work in conjunction withcreatine kinase now can be designed and used, individually, incombination or in addition to creatine compounds. Combinations ofcreatine compounds with other supplements or other drugs is proposed.

The pathways of biosynthesis and metabolism of creatine and creatinephosphate can be targeted in selecting and designing compounds which maymodify energy production or high energy phosphoryl transfer through thecreatine kinase system. Compounds targeted to specific steps may rely onstructural analogies with either creatine or its precursors. Novelcreatine analogs differing from creatine by substitution, chainextension, and/or cyclization may be designed. The substrates ofmultisubstrate enzymes may be covalently linked, or analogs which mimicportions of the different substrates may be designed. Non-hydrolyzablephosphorylated analogs can also be designed to mimic creatine phosphatewithout sustaining ATP production.

A number of creatine and creatine phosphate analogs have been previouslydescribed in the literature or can be readily synthesized. Examples arethese shown in Table 1 and Table 2. Some of them are slow substrates forcreatine kinase.

Tables 1 and 2 illustrate the structures of creatine, cyclocreatine(1-carboxymethyl-2-iminoimidazolidine), N-phosphorocreatine(N-phosphoryl creatine), cyclocreatine phosphate(3-phosphoryl-1-carboxymethyl-2-iminoimidazolidine) and other TABLE 1CREATINE ANALOGS

TABLE 2 CREATINE PHOSPHATE ANALOGS

compounds. In addition, 1-carboxymethyl-2-aminoimidazole,1-carboxymethyl-2 2-iminomethylimidazolidine,1-carboxyethyl-2-iminoimidazolidine, N-ethyl-N-amidinoglycine andb-guanidinopropionic acid are believed to be effective.

Cyclocreatine (1-carboxymethyl-2-iminoimidazolidine) is an example of aclass of substrate analogs of creatine kinase, which can bephosphorylated by creatine kinase and which are believed to be active.

A class of creatine kinase targeted compounds are bi-substrate analogscomprising an adenosine-like moiety linked via a modifiable bridge to acreatine link moiety (i.e., creatine or a creatine analog). Suchcompounds are expected to bind with greater affinity than the sum of thebinding interaction of each individual substrate (e.g., creatine andATP). The modifiable bridge linking an adenosine-like moiety at the5′-carbon to a creatine like moiety can be a carbonyl group, alkyl (abranched or straight chain hydrocarbon group having one or more carbonatoms), or substituted alkyl group (an alkyl group bearing one or morefunctionalities, including but not limited to unsaturation,heteroatom-substituents, carboxylic and inorganic acid derivatives, andelectrophilic moieties). Also the linking of creatine to other moleculesthat are energy enhancing or vitamins or antioxidants is recommended.Examples are creatine-pyruvate or creatine-ascorbate.

N-phosphorocreatine analogs also can be designed which bearnon-transferable moieties which mimic the N-phosphoryl group. Thesecannot sustain ATP production.

In one embodiment, the invention features methods of treating skindisorders by administering to a subject an effective amount of acreatine compound represented by the general formula I:

and pharmaceutically acceptable salts thereof, wherein:

a) Y is selected from the group consisting of: —CO₂H—NHOH, —NO₂, —SO₃H,—C(═O)NHSO₂J and —P(═O)(OH)(OJ), wherein J is selected from the groupconsisting of: hydrogen, C₁-C₆ straight chain alkyl, C₃-C₆ branchedalkyl, C₂-C₆ alkenyl, C₃-C₆ branched alkenyl, and aryl;

b) A is selected from the group consisting of: C, CH, C₁-C₅alkyl,C₂-C₅alkenyl, C₂-C₅alkynyl, and C₁-C₅alkoyl chain, each having 0-2substituents which are selected independently from the group consistingof:

-   -   1) K, where K is selected from the group consisting of: C₁-C₆        straight alkyl, C₂-C₆ straight alkenyl, C₁-C₆ straight alkoyl,        C₃-C₆ branched alkyl, C₃-C₆ branched alkenyl, and C₄-C₆ branched        alkoyl, K having 0-2 substituents independently selected from        the group consisting of rromo, chloro, epoxy and acetoxy;    -   2) an aryl group selected from the group consisting of: a 1-2        ring carbocycle and a 1-2 ring heterocycle, wherein the aryl        group contains 0-2 substituents independently selected from the        group consisting of: —CH₂L and —COCH₂L where L is independently        selected from the group consisting of: bromo, chloro, epoxy and        acetoxy; and    -   3) —NH-M, wherein M is selected from the group consisting of:        hydrogen, C₁-C₄ alkyl, C₂-C₄ alkenyl, C₁-C₄ alkoyl, C₃-C₄        branched alkyl, C₃-C₄ branched alkenyl, and C₄ branched alkoyl;

c) X is selected from the group consisting of NR₁, CHR₁, CR₁, O and S,wherein R₁ is selected from the group consisting of:

-   -   1) hydrogen;    -   2) K where K is selected from the group consisting of: C₁-C₆        straight alkyl, C₂-C₆ straight alkenyl, C₁-C₆ straight alkoyl,        C₃-C₆ branched alkyl, C₃-C₆ branched alkenyl, and C₄-C₆ branched        alkoyl, K having 0-2 substituents independently selected from        the group consisting of: bromo, chloro, epoxy and acetoxy;    -   3) an aryl group selected from the group consisting of a 1-2        ring carbocycle and a 1-2 ring heterocycle, wherein the aryl        group contains 0-2 substituents independently selected from the        group consisting of: —CH₂L and —COCH₂L where L is independently        selected from the group consisting of: bromo, chloro, epoxy and        acetoxy;    -   4) a C₅-C₉ a-amino-w-methyl-w-adenosylcarboxylic acid attached        via the w-methyl carbon;    -   5) 2 C₅-C₉ a-amino-w-aza-w-methyl-w-adenosylcarboxylic acid        attached via the w-methyl carbon; and    -   6) a C₅-C₉ a-amino-w-thia-w-methyl-w-adenosylcarboxylic acid        attached via the w-methyl carbon;

d) Z₁ and Z₂ are chosen independently from the group consisting of: ═O,—NHR₂, —CH₂R₂, —NR₂OH; wherein Z₁ and Z₂ may not both be =0 and whereinR₂ is selected from the group consisting of:

-   -   1) hydrogen;    -   2) K, where K is selected from the group consisting of: C₁-C₆        straight alkyl; C₂-C₆ straight alkenyl, C₁-C₆ straight alkoyl,        C₃-C₆ branched alkyl, C₃-C₆ branched alkenyl, and C₄-C₆ branched        alkoyl, K having 0-2 substituents independently selected from        the group consisting of bromo, chloro, epoxy and acetoxy;    -   3) an aryl group selected from the group consisting of a 1-2        ring carbocycle and a 1-2 ring heterocycle, wherein the aryl        group contains 0-2 substituents independently selected from the        group consisting of: —CH₂L and —COCH₂L where L is independently        selected from the group consisting of: bromo, chloro, epoxy and        acetoxy;    -   4) 2 C₄-C₉ a-amino-carboxylic acid attached via the w-carbon;    -   5) B, wherein B is selected from the group consisting of:        —CO₂H—NHOH, —SO₃H, —NO₂, OP(═O)(OH)(OJ) and —P(═O)(OH)(OJ),        wherein J is selected from the group consisting of: hydrogen,        C₁-C₆ straight alkyl, C₃-C₆ branched alkyl, C₂-C₆ alkenyl, C₃-C₆        branched alkenyl, and aryl, wherein B is optionally connected to        the nitrogen via a linker selected from the group consisting of:        C₁-C₂ alkyl, C₂ alkenyl, and C₁-C₂ alkoyl;    -   6) -D-E, wherein D is selected from the group consisting of:        C₁-C₃ straight alkyl, C₃ branched alkyl, C₂-C₃ straight alkenyl,        C₃ branched alkenyl, C₁-C₃ straight alkoyl, aryl and aroyl; and        E is selected from the group consisting of —(PO₃)_(n)NMP, where        n is 0-2 and NMP is ribonucleotide monophosphate connected via        the 5′-phosphate, 3′-phosphate or the aromatic ring of the base;        —[P(═O)(OCH₃)(O)]_(m)-Q, where m is 0-3 and Q is a        ribonucleoside connected via the ribose or the aromatic ring of        the base; —[P(═O)(OH)(CH₂)]_(m)-Q, where m is 0-3 and Q is a        ribonucleoside connected via the ribose or the aromatic ring of        the base; and an aryl group containing 0-3 substituents chosen        independently from the group consisting of: Cl, Br, epoxy,        acetoxy, —OG, —C(═O)G, and —CO₂G, where G is independently        selected from the group consisting of: C₁-C₆ straight alkyl,        C₂-C₆ straight alkenyl, C₁-C₆ straight alkoyl, C₃-C₆ branched        alkyl, C₃-C₆ branched alkenyl, C₄-C₆ branched alkoyl, wherein E        may be attached to any point to D, and if D is alkyl or alkenyl,        D may be connected at either or both ends by an amide linkage;        and    -   7)-E, wherein E is selected from the group consisting of        —(PO₃)_(n)NMP, where n is 0-2 and NMP is a ribonucleotide        monophosphate connected via the 5′-phosphate, 3′-phosphate or        the aromatic ring of the base; —[P(═O)(OCH₃)(O)]_(m)-Q, where m        is 0-3 and Q is a ribonucleoside connected via the ribose or the        aromatic ring of the base; —[P(═O)(OH)(CH₂)]_(m)-Q, where m is        0-3 and Q is a ribonucleoside connected via the ribose or the        aromatic ring of the base; and an aryl group containing 0-3        substituents chose independently from the group consisting of:        Cl, Br, epoxy, acetoxy, —OG, —C(═O)G, and —CO₂G, where G is        independently selected from the group consisting of: C₁-C₆        straight alkyl, C₂-C₆ straight alkenyl, C₁-C₆ straight alkoyl,        C₃-C₆ branched alkyl, C₃-C₆ branched alkenyl, C₄-C₆ branched        alkoyl; and if E is aryl, E may be connected by an amide        linkage;

e) if R₁ and at least one R₂ group are present, R₁ may be connected by asingle or double bond to an R₂ group to form a cycle of 5 to 7 members;

f) if two R₂ groups are present, they may be connected by a single or adouble bond to form a cycle of 4 to 7 members; and

g) if R₁ is present and Z₁ or Z₂ is selected from the group consistingof —NHR₂, —CH₂R₂ and —NR₂OH, then R₁ may be connected by a single ordouble bond to the carbon or nitrogen of either Z₁ or Z₂ to form a cycleof 4 to 7 members.

Creatine, creatine phosphate and many creatine analogs are commerciallyavailable. Additionally, analogs of creatine may be synthesized usingconventional techniques. For example, creatine can be used as thestarting material for synthesizing at least some of the analogsencompassed by formula I. Appropriate synthesis reagents, e.g.alkylating, alkenylating or alkynylating agents may be used to attachthe respective groups to target sites. Alternatively, reagents capableof inserting spacer groups may be used to alter the creatine structure.Sites other than the target site are protected using conventionalprotecting groups while the desired sites are being targeted bysynthetic reagents.

If the creatine analog contains a ring structure, then the analog may besynthesized in a manner analogous to that described for cyclocreatine(Wang, T., J. Org. Chem., 39:3591-3594 (1974)). The various othersubstituent groups may be introduced before or after the ring is formed.

Many creatine analogs have been previously synthesized and described(Rowley et al., J. Am. Chem. Soc. 93:5542-5551 (1971); McLaughlin etal., J. Biol. Chem. 247:4382-4388 (1972); Nguyen, A. C. K., “Synthesisand enzyme studies using creatine analogs”, Thesis, Dept. ofPharmaceutical Chemistry, Univ. Calif., San Francisco (1983); Lowe etal., J. Biol. Chem. 225:3944-3951 (1980); Roberts et al., J. Biol. Chem.260:13502-13508 (1985); Roberts et al., Arch. Biochem. Biophys.220:563-571 (1983), and Griffiths et al., J. Biol. Chem. 251:2049-2054(1976)). The contents of all of the aforementioned references areexpressly incorporated by reference. Further to the aforementionedreferences, Kaddurah-Daouk et al. (WO92/08456; WO90/09192; U.S. Pat. No.5,324,731; U.S. Pat. No. 5,321,030) also provide citations for thesynthesis of a plurality of creatine analogs. Also the synthesis ofcreatine-pyruvate and creatine ascorbate has been described in a seriesof patents WPI 98-481123/199841; WPI 98-457997/199840; WPI98-387651/199833). The contents of all the aforementioned references andpatents are incorporated herein by reference.

Creatine compounds which currently are available or have beensynthesized include, for example, creatine, b-guanidinopropionic acid,guanidinoacetic acid, creatine phosphate disodium salt, cyclocreatine,homocyclocreatine, phosphinic creatine, homocreatine, ethylcreatine,cyclocreatine phosphate dilithium salt and guanidinoacetic acidphosphate disodium salt, 4 guanidino benzoic acid and derivatives,creatine-pyruvate, creatine-ascorbate among others.

Creatine phosphate compounds also can be synthesized chemically orenzymatically. The chemical synthesis is well known. Annesley, T. M.Walker, J. B., Biochem. Biophys. Res. Commun., (1977), 74, 185-190;Cramer, F., Scheiffele, E., Vollmar, A., Chem. Ber., (1962), 95,1670-1682.

Salts of the products may be exchanged to other salts using standardprotocols. The enzymatic synthesis utilizes the creatine kinase enzyme,which is commercially available, to phosphorylate the creatinecompounds. ATP is required by creatine kinase for phosphorylation, henceit needs to be continuously replenished to drive the reaction forward.It is necessary to couple the creatine kinase reaction to anotherreaction that generates ATP to drive it forward. The purity of theresulting compounds can be confirmed using known analytical techniquesincluding ¹H NMR, ¹³CNMR Spectra, Thin layer chromatography, HPLC andelemental analysis.

Modes of Administration

The creatine compound can be administered to the afflicted individualalone or in combination with another creatine analog or other agent. Theother agents could be approved therapies, supplements that protectagainst oxidative damage, energy enhancers, sugars, intermediates ofmetabolism and nutrients among others. The creatine compounds can beadministered as pharmaceutically acceptable salts in a pharmaceuticallyacceptable carrier. The compound may be administered to the subject by avariety of routes, including, but not necessarily limited to topical,oral (dietary), transdermal, or parenteral (e.g., subcutaneous,intramuscular, intravenous injection, bolus or continuous infusion)routes of administration, for example. An effective amount (i.e., onethat is sufficient to produce the desired effect in an individual) of acomposition comprising a creatine analog is administered to theindividual. The actual amount of drug to be administered will depend onfactors such as the size and age of the individual, in addition to theseverity of symptoms, other medical conditions and the desired aim oftreatment. As discussed above, preferably the compound is administeredtopically.

Previous studies have described the administration and efficacy ofcreatine compounds in vivo. For example, creatine phosphate has beenadministered to patients with cardiac diseases by intravenous injection.Up to 8 grams/day were administered with no adverse side effects.Athletes and body builders take creatine monohydrate at 2-30 grams perday with an initial loading phase where 20 grams per day are usedfollowed by 2-5 grams per day to sustain levels. The efficacy ofselected creatine kinase substrate analogs to sustain ATP levels ordelay rigor during ischemic episodes in muscle has been investigated. Onone study, cyclocreatine was fed to mice, rats and chicks, and appearedto be well-tolerated in these animals. Newly hatched chicks were fed adiet containing 1% cyclocreatine. In the presence of antibiotics, thechicks tolerated 1% cyclocreatine without significant mortality,although the chicks grew more slowly than control chicks (Griffiths, G.R. and J. B. Walker, J. Biol. Chem. 251(7): 2049-2054 (1976)). Inanother study, mice were fed a diet containing 1% cyclocreatine for 10days (Annesley, T. M. and J. B. Walker, J. Biol. Chem. 253(22):8120-8125 (1978)). Cyclocreatine has been feed to mice at up to 1% oftheir diet for 2 weeks or for over 4 weeks without gross adverseeffects. Lillie et al., Cancer Res., 53: 3172-3178 (1993). Feedinganimals cyclocreatine (e.g., 1% dietary) has been shown to lead toaccumulation of cyclocreatine in different organs in mM concentrations.For example, cyclocreatine was reported to be taken up by muscle, heartand brain in rats receiving dietary 1% cyclocreatine. Griffiths, G. R.and J. B. Walker, J. Biol. Chem. 251(7): 2049-2054 (1976). As shownpreviously, antiviral activity of cyclocreatine is observed onadministering 1% dietary cyclocreatine. Many of the above-referencedstudies show that creatine analogs are been shown to be capable ofcrossing the blood-brain barrier. Creatine as 1%-3% of the diet wasshown to have beneficial effects on Huntington's disease.

The creatine compound can be formulated according to the selected routeof administration (e.g., emulsion, solution, cream, powder, tablet,capsule, transdermal patch, implantable capsule). An appropriatecomposition comprising a creatine analog can be prepared in aphysiologically acceptable vehicle or carrier. For example, acomposition in tablet form can include one or more additives such as afiller (e.g., lactose), a binder (e.g., gelatin, carboxymethylcellulose,gum arabic), a flavoring agent, a coloring agent, or coating material asdesired. For solutions or emulsions in general, carriers may includeaqueous or alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles can includesodium chloride, solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. In addition, intravenousvehicles can include fluid and nutrient replenishers, and electrolytereplenishers, such as those based on Ringer's dextrose. Preservativesand other additives can also be present. For example, antimicrobial,antioxidant, chelating agents, and inert gases can be added. (See,generally, Remington's Pharmaceutical Sciences, 16th Edition, Mack, Ed.,1980).

The term “administration” is intended to include routes ofadministration which allow the creatine compounds to perform theirintended function(s) of preventing, ameliorating, arresting, and/oreliminating disease(s) of the nervous system in a subject. Examples ofroutes of administration which may be used include injection (topical,oral, subcutaneous, intravenous, parenterally, intraperitoneally,inhalation, transdermal, and rectal. Depending on the route ofadministration, the creatine-like compound may be coated with or in amaterial to protect it from the natural conditions which maydetrimentally effect its ability to perform its intended function. Theadministration of the creatine-like compound is done at dosages and forperiods of time effective to reduce, ameliorate or eliminate thesymptoms of aging. Dosage regimes may be adjusted for purposes ofimproving the therapeutic or prophylacetic response of the compound. Forexample, several divided doses may be administered daily or the dose maybe proportionally reduced as indicated by the exigencies of thetherapeutic situation.

Exemplification of the Invention

1. Effects of 1% Creatine Supplementation on 3-nitrotyrosine/tyrosineConcentration in FALS Mice.

Oxidative injury involves the activation of nitric oxide production, andperoxynitrite which results in nitration of proteins. The nitration ofproteins could be determined by measuring the ratio of 3-nitrotryrosineto tyrosine. The FALS mice are transgenic animals that express a mutantform of Cu/Zn superoxide dismutase found in patients with familial ALS(Amyotrophic Lateral Sclerosis). These animals develop ALS symptoms withgradual motor neuron loss, muscle weakness, and die within 135 days.Oxidative stress has been associated with the death of motor neurons.Levels of 3-nitrotyrosine are significantly increased in the spinalcords of these mice (Ferrante 1997). The transgenic mice with the G93Amutation and the littermate controls (eight mice per group) were fed 1%creatine or unsupplemented diets at days 70 of age and then killed at120 days of age for measurements of 3-nitrotyrosine as described(Ferrante 1997). FIG. 1 (left panel) demonstrates that creatine cansignificantly inhibit the higher levels of 3 nitrotyrosine/tyrosinelevels in lower spinal cords of transgenic FALS. The right panel alsodemonstrates that creatine can inhibit the activation of production of3-nitrotyrosine/tyrosine in the upper spinal cords.

2. Effect of 1% Creatine Supplementation on Hydroxyl Radical Productionas Measured by Rate of Conversion of Salicylate to its by Products inFALS Mice.

The level of free radical production in vivo can be determined using themicrodialysis technique (Matthews et al. 1998). Administration of themitochondrial toxin 3-nitropropionic acid results in a significantincrease in the conversion of salicylate to 2,3-DHBA in the striatum,which is blocked in mice over expressing Cu, Zn SOD (Bogdanov et. al.,1998). Here we demonstrate that systemic administration of3-nitropropionic acid (3-NP) resulted in a significant increase in theconversion of 4-HBA to 3,4-DHBA in G93 A transgenic mice fedunsupplemented diets (FIG. 2). In animals fed 1% creatine supplementeddiets, there was no significant increase in 3,4 DHBA/4HBA after 3-NPadministration. This demonstrates that creatine can minimize theproduction of hydroxyl radicals that are implicated in aging relateddamage.

3. Production of 2,3 and 2,5 DHBA and 3 Nitrotyrosine (Indicators ofOxidative Stress) After Intrastriatal Injection of Malonate in ControlAnimals Fed with Creatine and Those Fed with Cyclocreatine

The salicylate hydroxyl radical-trapping method was used for measuringlevels of hydroxyl radicals in striatal tissue after malonateinjections. Eight animals in each group were fed either a normal diet ora diet enriched with 1% creatine or 1% cyclocreatine for two weeksbefore intrastriatal malonate injections. Animals were injected with 200mg/kg salicylate intraperitoneally just before the malonate injectionsand were killed 1 hour later. The striata were then dissected rapidlyfrom a 2-mm thick slice and placed in 0.25 ml of chilled 0.1 Mperchloric acid. Samples were subsequently sonicated, frozen rapidly andthawed and centrifuged twice. An aliquot of supernatant was analyzed byHPLC with the 16-electrode electrochemical detection (Beal et. al.,1990). Salicylate, 2,3 and 2,5 DHBA, tyrosine, 3-nitrotyrosine weremeasured electrochemically by oxidation at 840, 240, 120, 600 and 840 mVrespectively with retention times of 20.5, 9.4, 6.3, 10.5, 18.2 minrespectively. The data were expressed as the ratio of 2, 3 and 2,5 DHBAto salicylate to normalize the DHBA concentrations for differing brainconcentrations of salicylate. Similarly, 3-nitrotyrosine levels werenormalized to tyrosine levels. We also examined the effects of 1%creatine supplementation for 2 weeks on 3-NP induced increases in3-nitrotyrosine levels. Male Sprague Dawley rats were treated with 3-NPat a dose of 20 mg/kg intraperotoneally and then killed at 3 hours. Tenanimals were examined in each group. The striata were dissected andplaced in chilled 0.1 M perchloric acid. 3-Nitrotyrosine and tyrosineconcentrations were measured by HPLC with electrochemical detection(Matthews 1998). Statistical comparisons were made by unpaired Student'st test or by one way ANOVA followed by Fisher's protected leastsignificant difference test for post hoc comparisons.

FIG. 3 demonstrates that both creatine and cyclocreatine can protectagainst increases in levels of salisylate derivatives 2,3 DHBA and 2,5DHBA after injection of the mitochondrial toxin malonate. This confirmsthat creatine compounds can indeed protect against production ofhydroxyl radicals implicated in oxidative stress and mitochondrialdysfunction. Also FIG. 3 demonstrates that creatine and cyclocreatinehave protective effects against nitration of proteins induced by themitochondrial toxin malonate. Production of nitric oxide andperoxynitrite are part of the cascade of oxidative damage. FIG. 4illustrates the protective effect of creatine against nitration ofproteins induced by another mitochondrial toxin 3-NP.

The conclusion of the experimental results discussed above confirms theimportance of the function of the creatine kinase system and thecreatine compounds in protecting against cascades of oxidative stress.The process of aging is believed to involve mitochondrial dysfunctionand oxidative damage resulting from the production of molecules likehydroxyl radicals, nitric oxide and peroxynitrite. Our results stronglysuggest that creatine compounds could indeed minimize damage inducedduring the process of aging.

Utility

In the present invention, the creatine compounds can be administered toan individual (e.g., a mammal), alone or in combination with anothercompound, for the prevention or treatment of aging or insult relateddamage to skin. As agents for skin preservation, creatine compounds canmodify creatine kinase/phosphocreatine functions, energy state,oxidative damage, and/or cell survival thereby preventing, ameliorating,arresting or eliminating direct and/or indirect effects of skin damageduring aging, exposure to UV radiation, stress, fatigue or otherinsults. Other compounds which can be administered together with thecreatine compounds include antioxidants, vitamins, energy enhancingagents, and other agents used for skin care.

A variety of skin diseases can be treated with creatine or creatineanalogs, including but not limited to damage induced during aging orharmful sun radiations. Creatine or analogs of creatine can be used toreduce the severity of damage or inhibit its development. Creatine,creatine phosphate or analogs such as 3-guanidinopropionic acid,guanidino acetate, creatine-pyruvate, creatine ascorbate can be used totreat age related skin damage.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

The entire contents of all references, patents, and patent applicationscited herein are expressly incorporated by reference.

1. A method for treating un even pigmentation of the skin, comprisingadministering an effective amount of a creatine compound to a subject,such that said subject is treated for uneven pigmentation of the skin.2. The method of claims 1, wherein said creatine compound isadministered topically or orally.
 3. The method of claim 1, wherein saidsubject is a human.
 4. The method of claim 1, wherein said creatinecompound is creatine, creatine citrate, creatine ascorbate,cyclocreatine, or creatine phosphate.
 5. The method of claim 1, furthercomprising administering carnitine or CoQ10.
 6. The method of claim 1,wherein said creatine compound is administered in combination with apharmaceutically acceptable carrier.
 7. The method of claim 6, whereinsaid pharmaceutically acceptable carrier is suitable for administerationas a lotion, cream, mousse, aerosol, gel, emulsion, solution, ointment,or medicated pad.
 8. A pharmaceutical composition, comprising aneffective amount of a creatine compound and a pharmaceuticallyacceptable carrier, wherein said effective amount is effective fortreating uneven pigmentation of the skin.
 9. The pharmaceuticalcomposition of claim 8, wherein said pharmaceutically acceptable carrieris suitable for topical or oral administration.
 10. The pharmaceuticalcomposition of claim 8, wherein said pharmaceutically acceptable carrieris suitable for adminsisteration as lotion, cream, mousse, aerosol, gel,emulsion, solution, ointment, or medicated pad.
 11. The pharmaceuticalcomposition of claim 8, wherein said creatine compound is creatine,creatine citrate, creatine ascorbate, cyclocreatine, or creatinephosphate.